The goal of the proposed research is to use the fruit fly, Drosophila melanogaster, as an animal model to unravel the molecular mechanisms underlying the biosynthesis, turnover and non-classical functions of rhodopsins. Rhodopsin is comprised of an opsin protein and a vitamin A-derived chromophore, which senses light. Among the most common forms of retinal degeneration are those that result from defects in the visual cycle (retinoid cycle)-an enzymatic pathway required for regeneration of the chromophore. Until recently it was thought that flies do not employ a visual cycle, since the chromophore does not normally release from photoactivated rhodopsin. However, some rhodopsin is internalized and the opsin gets degraded, thereby releasing the chromophore. During the last funding period, we made the discovery that flies use a visual cycle to regenerate the released chromophore. The experiments proposed in aim 1 are designed to differentiate between competing hypotheses to explain the basis for the retinal degeneration that results from defects in this enzymatic pathway. We also propose experiments to test hypotheses predicted by the newly formulated model for the invertebrate cycle. Since the mammalian opsin (melanopsin) that functions in the intrinsically photosensitive retinal ganglion cells appears to be more akin to Drosophila rhodopsins than to rod and cone photopigments, these studies also suggest that a visual cycle might function to regenerate the chromophore used by melanopsin. Although some rhodopsin is normally internalized, excessive internalization and degradation of rhodopsin occurs in a variety of flies with mutations that hyperactivate the phototransduction cascade. This appears to be a feedback mechanism to limit excessive signaling. The second aim addresses a new hypothesis that would explain how uncontrolled activity of the heterotrimeric G-protein leads to excessive turnover of rhodopsin. Although rhodopsins that function in the retina are among the best-characterized receptor proteins, in the past few years it has become clear that some opsins are expressed outside the retina. However, their extra-retinal roles are understood poorly. Aims 3 and 4 of the proposed research will characterize the roles of two opsins that are expressed in neurons in the olfactory system and central brain and that have not been associated with a light response. We propose to test the contributions of these rhodopsins to animal behaviors. The proposed experiments raise intriguing possibilities as to the potential roles for mammalian extra-retinal opsins, such as Opn3 and Opn5, which have not been subjected to genetic analysis. To accomplish our goals, we propose to employ a multidisciplinary approach using a combination of genetic, cell biological, electrophysiological, molecular and biochemical techniques. The long-term goals of these studies are to 1) uncover mechanisms underlying the retinal degenerations that result from defects in the visual cycle with the ultimate goal of discovering new therapeutic approaches, and 2) uncover the roles of the enigmatic extra-retinal opsins. PUBLIC HEALTH RELEVANCE: Rhodopsin is the receptor that is critically important for detecting light, and mutations affecting rhodopsin lead to common forms of retinal degeneration. The focus of the proposed work is to exploit the great technical advantages of the fruit fly as an animal model to uncover mechanisms underlying the retinal degeneration resulting from impairments in rhodopsins, and to identify roles for opsins that are expressed in cells that are not known to function in light reception.